Study on the Efficacy and Mechanism of Lycium barbarum Polysaccharide against Lead-Induced Renal Injury in Mice

Lead is one of the most common heavy metal pollutants in the environment. Prolonged exposure to lead will induce oxidative stress, inflammation, and apoptosis in the kidneys, which in turn causes kidney injury. Lycium barbarum polysaccharide (LBP) is well known for its numerous pharmacological properties. This study aims to explore the efficacy and mechanism of LBP against lead-induced kidney damage in mice. Symptoms of renal injury were induced in mice by using 25 mg/kg lead acetate (PbAc2), and different doses of LBP (200, 400, and 600 mg/kg BW) were orally administrated to PbAc2-treated mice for five weeks. The results of the pharmacodynamics experiment showed that the renal pathological damages, serum creatinine (Cre), blood urea nitrogen (BUN), and kidney index of PbAc2-treated mice could be significantly alleviated by treatment with LBP. Further, LBP treatment significantly increased the weight and feed intake of PbAc2-treated mice. The dose effect results indicated that a medium dose of LBP was superior to high and low doses. The results of mechanistic experiments showed that LBP could attenuate oxidative stress, inflammation, and apoptosis in the kidneys of mice with lead toxicity by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.


Introduction
Lead is considered one of the most polluting elements in the world and carries high non-carcinogenic risks for children and adults [1,2]. Lead pollution in the environment mainly comes from lead-zinc ore smelting, non-ferrous metal polymetallic ore smelting, electronic waste recovery, and leaded gasoline consumption [3]. Lead can be absorbed by humans via the alimentary tract, respiratory tract, and skin [4]. After intake of lead, it firstly enters the bloodstream, and during circulation, toxic metals are distributed to the kidneys, causing kidney damage [5].
The mechanisms underlying renal injury due to lead toxicity are multifaceted. Studies have shown that lead exposure induces renal inflammation and increases the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the kidney tissue [6]. Furthermore, lead exposure can promote an oxidative stress response, significantly increase the level of malondialdehyde (MDA) in renal tissue, and reduce the levels of superoxide dismutase (SOD), glutathione (GSH), and glutathione S-transferase (GST) in the tissue, which in turn induces the excessive production of reactive oxygen species (ROS) and stimulates an apoptotic cascade reaction, leading to the release of cytochrome-C and the activation of the caspase (Casp) enzyme. At the same time, it

Blood and Kidney Tissue Collection
The mice were anesthetized, and blood was collected orbitally in 1.5 mL, clean sterile centrifuge tubes, then immediately centrifuged (3000 r/min, 10 min) before serum was collected and stored in a −20 • C freezer for later use. The mice were sacrificed by cervical dislocation; the kidneys were then immediately removed, and the bloodstain was rinsed with pre-cooled saline. After blotting on filter paper, the mass was weighed with a precision electronic balance; the left kidney was immediately immersed in the configured formalin solution for further use, and the right kidney was preserved in a −80 • C freezer for further use. The mice were weighed every seven days, and the average weight of the mice in each group was calculated. Each group was fed with 70 g feed every day (making sure there was a daily surplus of feed), and the remaining feed was weighed at 9 am the next day to calculate the daily feed intake of each group.

Measurement of Renal Index
The kidney was weighed, using the following formula: Organ weight index = [Wet weight of organ (g)/Bodyweight (g)] × 100.

Detection of Renal Function Biomarkers
After thawing the serum, the expression levels of Cre and BUN in the mouse serum were measured by ELISA. After sequential addition of samples, standards, antibodies, and color development agents according to the procedure of the kit, the concentrations of Cre and BUN in the mouse serum were calculated by measuring the absorbance (OD value) at a 450 nm wavelength using a microplate reader (model number: Max Plus 384).

Histopathological Examination
Formalin solution (10% formaldehyde in distilled water) was used to fix the kidney samples paraffin embedding. The kidney samples were stained with hematoxylin-eosin (H&E) and 4 µm sections were cut for histopathological evaluations. Images were captured at a 400 times magnification. Pathological changes of the glomerulus, renal tubular injury, inflammation were graded as mild (+), moderate (++), or severe (+++) in each group.

Study on the Mechanism of LBP against Lead-Induced Renal Injury in Mice 2.5.1. Detection of Oxidative Stress Markers in Renal Tissue
We took 0.1 g renal tissue, added 0.9 mL normal saline, made a 10% homogenate of the renal tissue, centrifuged (3000 r/min, 10 min), and took the supernatant. The expression levels of SOD, MDA, GSH, and GST were detected by ELISA. After sequential addition of samples, standards, antibodies, and color development agents according to the procedure of the kit, the concentrations of SOD, MDA, GSH, and GST in the mouse serum were calculated by measuring the absorbance (OD value) at a 450 nm wavelength using a microplate reader.

Detection of Renal Inflammatory Biomarkers
The IL-1β, TNF-α, and NO levels in the renal tissue were determined using ELISA kits according to the manufacturer's guidelines (consistent with 2.5.1 operation steps).

Detection of Apoptotic Biomarkers
The Cas-3, Bax, and Bcl-2 levels in the renal tissue were determined using ELISA kits according to the manufacturer's guidelines (consistent with 2.5.1 operation steps).

Western Blotting Analyses
After thawing the kidney tissue of each group of mice, 40 mg of homogenate was taken, and the total protein was extracted with the BCA kit to determine the protein content. We extracted the same amount of protein sample and denatured it at 100 • C for 5 min. We used the sodium lauryl sulfate-polyacrylamide gel electrophoresis method to separate the protein and transfer it to a polyvinylidene fluoride membrane, where we added the corresponding primary antibody at 4 • C and incubated overnight, before washing and adding horseradish peroxide at 4 • C. The enzyme-labeled secondary antibody was incubated for 2 h; then, we added luminescent solution after washing, and exposed [11]. We used ImageJ to count the gray values.

Statistical Analysis
Data analysis was carried out using the Statistical Package for Social Sciences (SPSS version 26) software. The collected data were analyzed using one-way analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant. All values were expressed as the mean ± standard deviation (SD) of the mean.

Dose-Effect Analysis
Orthogonal Partial least squares discriminant analysis (OPLS-DA) is often used to deal with classification and discriminant problems. It was similar to principal component analysis (PCA) except that PCA was unsupervised and OPLS-DA was supervised [32]. In order to visualize the dose-effect relationship of LBP against lead-induced renal injury, SIMCA-P14.1 software was used for multivariate statistical analysis. The expression levels of oxidative stress markers (SOD, MDA, GSH, GST), inflammatory markers (TNF-α, IL-1β, NO), and apoptotic markers (Casp-3, Bax, Bcl-2) were used as variables to establish an unsupervised PCA model and a supervised OPLS-DA model. Finally, Oebiotech software (available online: https://cloud.oebiotech.cn/task/website (accessed on 16 July 2021)) was used to analyze the efficacy index and mechanism index by cluster heat map, and the global expression change of multisample and multivariable is presented intuitively.

Effect of LBP on Body Weight
There was no significant difference in the initial body weights of the mice in each group (Table 1). During the experiment, the model group had the slowest growth rate, the middle-dose group had the fastest growth rate, and the high-dose, low-dose, and control groups had no significant differences. After the experiment, the average body weight of mice in the model group was significantly lower than that of the control group (p < 0.05), while that of the LBP administration group was significantly higher than that of the model group (p < 0.01) with no significant difference from the control group (p ≥ 0.05). These findings are shown in Figure 1a. Table 1. Initial weight and final weight as the mean ± SD (n = 10).

Group
Initial There was no significant difference in the initial body weights of the mice in each group (Table 1). During the experiment, the model group had the slowest growth rate, the middle-dose group had the fastest growth rate, and the high-dose, low-dose, and control groups had no significant differences. After the experiment, the average body weight of mice in the model group was significantly lower than that of the control group (p < 0.05), while that of the LBP administration group was significantly higher than that of the model group (p < 0.01) with no significant difference from the control group (p ≥ 0.05). These findings are shown in Figure 1a. Table 1. Initial weight and final weight as the mean ± SD (n = 10).

Group
Initial

Effect of LBP on Feed Intake
The feed intake of mice in the model group was significantly decreased compared with the control group (p < 0.01). In the LBP administration group, the feed intake of mice significantly increased (p < 0.01) and there was no significant difference with the control group (p > 0.05). These findings are shown in Figure 1b.

Effect of LBP on Kidney Index
Compared with the control group, the renal index was significantly higher in the model group (p < 0.01). The low-dose and high-dose groups showed a significant decreasing trend compared with the model group (p < 0.05), and the middle-dose group showed an extremely significant decreasing trend (p < 0.01). These findings are shown in Figure 1c.

Effect of LBP on Renal Function Biomarkers
Compared with the control group, the expression levels of BUN ( Figure 1d) and Cre (Figure 1e) in the model group were significantly increased (p < 0.01). The LBP administration group had significantly reduced expression levels of BUN and Cre (p < 0.01). The medium-dose group had downregulated the levels of BUN and Cre better than the high-dose and low-dose groups.

Effects of LBP on Histopathology
A normal structure of the renal tissue without histopathological deformations was observed in the control mice ( Figure 2a). However, the PbAc 2 -exposed mice had a shrunken and congested glomerulus, widened urinary space, inflammatory cells in the interstitial tissue, and damaged renal tubules with vacuolated cytoplasm (Figure 2b). In contrast, LBP pretreatment alleviated the changes induced in the renal tubules and glomeruli by PbAc 2 . These effects are shown in Table 2.

Effect of LBP on Feed Intake
The feed intake of mice in the model group was significantly decreased compared with the control group (p < 0.01). In the LBP administration group, the feed intake of mice significantly increased (p < 0.01) and there was no significant difference with the control group (p > 0.05). These findings are shown in Figure 1b.

Effect of LBP on Kidney Index
Compared with the control group, the renal index was significantly higher in the model group (p < 0.01). The low-dose and high-dose groups showed a significant decreasing trend compared with the model group (p < 0.05), and the middle-dose group showed an extremely significant decreasing trend (p < 0.01). These findings are shown in Figure 1c.

Effect of LBP on Renal Function Biomarkers
Compared with the control group, the expression levels of BUN ( Figure 1d) and Cre (Figure 1e) in the model group were significantly increased (p < 0.01). The LBP administration group had significantly reduced expression levels of BUN and Cre (p < 0.01). The medium-dose group had downregulated the levels of BUN and Cre better than the highdose and low-dose groups.

Effects of LBP on Histopathology
A normal structure of the renal tissue without histopathological deformations was observed in the control mice ( Figure 2a). However, the PbAc2-exposed mice had a shrunken and congested glomerulus, widened urinary space, inflammatory cells in the interstitial tissue, and damaged renal tubules with vacuolated cytoplasm (Figure 2b). In contrast, LBP pretreatment alleviated the changes induced in the renal tubules and glomeruli by PbAc2. These effects are shown in Table 2.

Effects of LBP on Oxidative Stress Markers
The results showed that lead acetate significantly decreased the levels of SOD (

Effects of LBP on Renal Inflammatory Biomarkers
Compared with the control group, the concentrations of IL-1β (Figure 4b), TNF-α (Figure 4a), and NO ( Figure 4c) in the model group were increased and showed significant differences (p < 0.01). Compared with the model group, the middle-dose group of LBP could downregulate the IL-1β, TNF-α, and NO expression levels, with statistical differences (p < 0.01). The high-dose group could significantly downregulate the expression levels of IL-1β and TNF-α (p < 0.01), along with the expression level of NO (p < 0.05). The LBP low-dose group could significantly downregulate the expression levels of TNF-α and IL-1β with a statistical difference (p < 0.01), but the downregulation of NO was not statistically different (p > 0.05).

Effects of LBP on Renal Inflammatory Biomarkers
Compared with the control group, the concentrations of IL-1β (Figure 4b), TNF-α (Figure 4a), and NO ( Figure 4c) in the model group were increased and showed significant differences (p < 0.01). Compared with the model group, the middle-dose group of LBP could downregulate the IL-1β, TNF-α, and NO expression levels, with statistical differences (p < 0.01). The high-dose group could significantly downregulate the expression levels of IL-1β and TNF-α (p < 0.01), along with the expression level of NO (p < 0.05). The LBP low-dose group could significantly downregulate the expression levels of TNF-α and IL-1β with a statistical difference (p < 0.01), but the downregulation of NO was not statistically different (p > 0.05).

Effects of LBP on Apoptotic Biomarkers
Compared with the control group, the expression levels of Casp-3 ( Figure 5a) and Bax ( Figure 5b) in the model group were significantly increased (p < 0.01), while the expression of Bcl-2 ( Figure 5c) was significantly decreased (p < 0.01). The LBP administration group significantly downregulated Casp-3 and Bax protein expression (p < 0.01), and upregulated Bcl-2 protein expression (p < 0.01).

Effects of LBP on Apoptotic Biomarkers
Compared with the control group, the expression levels of Casp-3 ( Figure 5a) and Bax (Figure 5b) in the model group were significantly increased (p < 0.01), while the expression of Bcl-2 (Figure 5c) was significantly decreased (p < 0.01). The LBP administration group significantly downregulated Casp-3 and Bax protein expression (p < 0.01), and upregulated Bcl-2 protein expression (p < 0.01).

Effects of LBP on Renal Inflammatory Biomarkers
Compared with the control group, the concentrations of IL-1β (Figure 4b), TNF-α (Figure 4a), and NO (Figure 4c) in the model group were increased and showed significant differences (p < 0.01). Compared with the model group, the middle-dose group of LBP could downregulate the IL-1β, TNF-α, and NO expression levels, with statistical differences (p < 0.01). The high-dose group could significantly downregulate the expression levels of IL-1β and TNF-α (p < 0.01), along with the expression level of NO (p < 0.05). The LBP low-dose group could significantly downregulate the expression levels of TNF-α and IL-1β with a statistical difference (p < 0.01), but the downregulation of NO was not statistically different (p > 0.05).

Expression of Proteins Related to the Nrf2 Signaling Pathway
To study the signal pathway of LBP against lead-induced renal injury, the expression of Nrf2 signal pathway-related proteins was detected (Figure 6a). The results showed that compared with the control group, the protein expression levels of Nrf2 ( Figure 6b) and Keap1 (Figure 6c) in the model group were significantly decreased (p < 0.01). Further, the protein expression of HO-1 (Figure 6d) was significantly increased (p < 0.01) and the protein expression of NQO1 (Figure 6e) was not statistically different (p > 0.05). Compared with the model group, the protein expression levels of Nrf2, HO-1, and NQO1 in the LBP administration group were significantly increased (p < 0.01), with the most significant trend observed in the middle-dosage group. All of the Keap1 protein expression levels were significantly decreased (p < 0.01). These results indicated that lead could lead to disruption of the antioxidant system by depleting the Nrf2 and Keap1 proteins in vivo, while LBP could activate the Nrf2 signaling pathway and promote the production of antioxidant enzymes protecting against lead-induced kidney injury, with the medium dose having the best effect. To study the signal pathway of LBP against lead-induced renal injury, the expression of Nrf2 signal pathway-related proteins was detected (Figure 6a). The results showed that compared with the control group, the protein expression levels of Nrf2 ( Figure 6b) and Keap1 (Figure 6c) in the model group were significantly decreased (p < 0.01). Further, the protein expression of HO-1 (Figure 6d) was significantly increased (p < 0.01) and the protein expression of NQO1 (Figure 6e) was not statistically different (p > 0.05). Compared with the model group, the protein expression levels of Nrf2, HO-1, and NQO1 in the LBP administration group were significantly increased (p < 0.01), with the most significant trend observed in the middle-dosage group. All of the Keap1 protein expression levels were significantly decreased (p < 0.01). These results indicated that lead could lead to disruption of the antioxidant system by depleting the Nrf2 and Keap1 proteins in vivo, while LBP could activate the Nrf2 signaling pathway and promote the production of antioxidant enzymes protecting against lead-induced kidney injury, with the medium dose having the best effect.

Multivariate Statistics
The PCA results are shown in (Figure 7a). The R2X was 0.794 and Q2 was 0.586, indicating that the PCA model had good interpretation and prediction ability. There was an obvious separation trend between the control group and the model group in PCA score plots, which indicated that lead could induce renal injury in mice through oxidative stress and inflammatory and apoptotic responses. The position of mice in the PCA score plot of the LBP administration group was close to the control group. Mice in the middle-dose group were closer to the control group, which indicated that LBP could protect mice from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptosis, with the middle-dose group having the best efficacy.
To further show the variation trend among the groups, OPLS-DA model results showed that the R2X was 0.994, R2Y was 0.23, and Q2 of 200 permutation validation was 21.1%, confirming that this OPLS-DA model had a good fit and predictive ability ( Figure  7b). Furthermore, the intercept of the Q2 regression line was −0.123 (Figure 7c), indicating that the model was not overfitted and was statistically significant. Based on these results, the OPLS-DA score showed consistent results with the PCA model, which further illustrated that LBP could protect from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptotic responses. The efficacy was better at the middle dose, followed by the high dose, and finally the low dose.

Multivariate Statistics
The PCA results are shown in (Figure 7a). The R2X was 0.794 and Q2 was 0.586, indicating that the PCA model had good interpretation and prediction ability. There was an obvious separation trend between the control group and the model group in PCA score plots, which indicated that lead could induce renal injury in mice through oxidative stress and inflammatory and apoptotic responses. The position of mice in the PCA score plot of the LBP administration group was close to the control group. Mice in the middle-dose group were closer to the control group, which indicated that LBP could protect mice from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptosis, with the middle-dose group having the best efficacy.

Multivariate Statistics
The PCA results are shown in (Figure 7a). The R2X was 0.794 and Q2 was 0.586, indicating that the PCA model had good interpretation and prediction ability. There was an obvious separation trend between the control group and the model group in PCA score plots, which indicated that lead could induce renal injury in mice through oxidative stress and inflammatory and apoptotic responses. The position of mice in the PCA score plot of the LBP administration group was close to the control group. Mice in the middle-dose group were closer to the control group, which indicated that LBP could protect mice from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptosis, with the middle-dose group having the best efficacy.
To further show the variation trend among the groups, OPLS-DA model results showed that the R2X was 0.994, R2Y was 0.23, and Q2 of 200 permutation validation was 21.1%, confirming that this OPLS-DA model had a good fit and predictive ability ( Figure  7b). Furthermore, the intercept of the Q2 regression line was −0.123 (Figure 7c), indicating that the model was not overfitted and was statistically significant. Based on these results, the OPLS-DA score showed consistent results with the PCA model, which further illustrated that LBP could protect from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptotic responses. The efficacy was better at the middle dose, followed by the high dose, and finally the low dose.  To further show the variation trend among the groups, OPLS-DA model results showed that the R2X was 0.994, R2Y was 0.23, and Q2 of 200 permutation validation was 21.1%, confirming that this OPLS-DA model had a good fit and predictive ability (Figure 7b). Furthermore, the intercept of the Q2 regression line was −0.123 (Figure 7c), indicating that the model was not overfitted and was statistically significant. Based on these results, the OPLS-DA score showed consistent results with the PCA model, which further illustrated that LBP could protect from lead-induced renal injury by inhibiting oxidative stress, inflammation, and apoptotic responses. The efficacy was better at the middle dose, followed by the high dose, and finally the low dose.

Cluster Heatmap Analysis
The results of the cluster heatmap showed that compared with the control group, the indexes of body weight, SOD, GST, GSH, and Bcl-2 in the model group were downregulated, while the NO, kidney index, TNF-α, IL-1β, MDA, Bax, BUN, case-3, and Cre were upregulated. Compared with the model group, the LBP administration groups all presented parameters that reversed the above indicators, and all showed a tendency to recover towards the control group, with the middle dose the closest to the control group. This suggests that LBP can inhibit lead-induced kidney damage through the antioxidant, anti-inflammatory, and anti-apoptotic pathways, with the best effect at medium doses ( Figure 8).

Cluster Heatmap Analysis
The results of the cluster heatmap showed that compared with the control group, the indexes of body weight, SOD, GST, GSH, and Bcl-2 in the model group were downregulated, while the NO, kidney index, TNF-α, IL-1β, MDA, Bax, BUN, case-3, and Cre were upregulated. Compared with the model group, the LBP administration groups all presented parameters that reversed the above indicators, and all showed a tendency to recover towards the control group, with the middle dose the closest to the control group. This suggests that LBP can inhibit lead-induced kidney damage through the antioxidant, anti-inflammatory, and anti-apoptotic pathways, with the best effect at medium doses ( Figure 8).

Discussion
It is public knowledge that lead exposure or lead poisoning can cause a series of physiological, biochemical, and behavioral disorders in experimental animals and humans [33][34][35].
Our study showed that the growth rate of mice was significantly slower, and it significantly reduced the body weight of mice after lead exposure (Figure 1a), which was consistent with previous findings [36]. However, supplementation of LBP to PbAc2treated mice significantly improved the growth rate and average daily feed intake ( Figure  1b), and it was able to increase the weights of the mice (Table 1). Early studies have shown that lead exposure causes the body to be immunosuppressed, which increases disease susceptibility [37], and LBP is considered to be a good source of potential prebiotics that can enhance the gut microbiota, increase the level of beneficial bacteria, and regulate innate immune responses [38]. Chen [39] et al.'s study showed that LBP could significantly increase the average daily weight and daily feed intake of weaned piglets. The mechanism may be through enhancing the body's immune status and antioxidant capacity and improving the intestinal microbial population. It is suggested that LBP may enhance the immunity and antioxidant capacity of the body, improve the number of intestinal microflora, enhance the appetite of lead poisoned mice, and promote the growth of mice.
A large number of studies have shown that lead exposure not only produces weight loss in mice but also induces multiple organ injuries in humans. When exposed to lead, the kidney is one of the most affected organs [40]. Our results showed that the kidney

Discussion
It is public knowledge that lead exposure or lead poisoning can cause a series of physiological, biochemical, and behavioral disorders in experimental animals and humans [33][34][35].
Our study showed that the growth rate of mice was significantly slower, and it significantly reduced the body weight of mice after lead exposure (Figure 1a), which was consistent with previous findings [36]. However, supplementation of LBP to PbAc2-treated mice significantly improved the growth rate and average daily feed intake (Figure 1b), and it was able to increase the weights of the mice (Table 1). Early studies have shown that lead exposure causes the body to be immunosuppressed, which increases disease susceptibility [37], and LBP is considered to be a good source of potential prebiotics that can enhance the gut microbiota, increase the level of beneficial bacteria, and regulate innate immune responses [38]. Chen [39] et al.'s study showed that LBP could significantly increase the average daily weight and daily feed intake of weaned piglets. The mechanism may be through enhancing the body's immune status and antioxidant capacity and improving the intestinal microbial population. It is suggested that LBP may enhance the immunity and antioxidant capacity of the body, improve the number of intestinal microflora, enhance the appetite of lead poisoned mice, and promote the growth of mice.
A large number of studies have shown that lead exposure not only produces weight loss in mice but also induces multiple organ injuries in humans. When exposed to lead, the kidney is one of the most affected organs [40]. Our results showed that the kidney index was significantly increased in the model group compared with the control group (Figure 1c), which was consistent with previous reports [7], and LBP administration reduced the kidney index of mice with lead toxicity. Lead-induced kidney injury was also confirmed by the observation of renal pathological sections (Figure 2), which showed obvious early-stage proximal tubule vacuolization, tissue inflammation, and tubule dilatation in the kidneys of model mice compared with controls, whereas the mice in the LBP administration group all improved in terms of the above symptoms. Researchers found that some natural compounds can reduce lead acetate-induced nephrotoxicity through antioxidant, anti-inflammatory, and anti-apoptotic activities [6,41]. LBP has been proved to possess biological properties such as antioxidant, anti-inflammation, and anti-apoptosis properties in different models, tissues, and organs. Huang et al. [30] found that LBP could reduce inflammation and activate antioxidant responses to protect from kidney injury in septic rats by regulating proinflammatory cytokine levels and the Keap1-Nrf2/antioxidant response element(ARE) signaling pathways. Xiao et al.'s [42] experiment showed that LBP alleviated ethanol-induced liver injury by reducing hepatic apoptosis, oxidative stress, and NOD-like receptor 3 (NLRP3) inflammasome.
BUN and Cre levels are commonly used to reflect renal function status. Both are nitrogen-containing endproducts of metabolism; BUN is the primary metabolite derived from dietary protein and tissue protein turnover, and Cre is a product of muscle creatine catabolism [43]. Our study showed that the levels of BUN (Figure 1d) and Cre (Figure 1e) were significantly increased in the model mice compared with the control group, which indicated that our modeling was successful and that the LBP-administrated group exhibited significantly lower levels of BUN and Cre than the model group, suggesting that LBP may have blunted the development of renal injury. According to Zhao et al. [44], LBP can improve the renal function of diabetic rats and reduce kidney damage by reducing oxidative stress and inhibiting the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in mesangial cells. Du et al. [45]. believed that the mechanism of LBP protecting the kidneys of diabetic rats was the inhibition of proteinuria, the blood urea nitrogen concentration, and serum inflammatory factors (including IL-2, IL-6, and TNF-α). Yu et al.'s [46] study showed that LBP has renoprotective effects, which may protect mice from hyperuricemia by promoting renal uric acid excretion. Liao et al. [47] suggested that the mechanism of LBP against high-fat diet-induced renal injury in rats may be to mediate lipid metabolism, enhance anti-inflammatory responses, and ameliorate renal injury caused by lipid metabolism regulators.
Based on the above conclusions, the mechanism of LBP's protection against leadinduced renal injury was studied. It is well-known that the mechanism of lead-induced kidney injury is multifaceted. Lead can stop the synthesis of structural proteins by binding to sulfhydryl proteins, thereby reducing the reserve of the organism's sulfhydryl antioxidants, and eventually causing damage to the organism by depleting the body's antioxidants [48][49][50]. GSH is an intracellular non-enzymatic antioxidant-containing thiols that have antioxidant and detoxification effects [51]. Therefore, lead can inhibit the activity of antioxidant GSH by inhibiting the effect of thiols [52]. As mentioned in the article by Veena Sharma et al. [53], peroxyl radicals are generated after lead poisoning, endoperoxides are generated through cyclization reaction, lipid peroxidation is stimulated, and the increased level of MDA indicates enhanced lipid peroxidation. Lead can induce oxidative stress (OS) by producing reactive oxygen species (ROS). The production of reactive oxygen species exceeds the ability of the antioxidant system to protect cells from oxidative molecules, and so oxidative stress will occur [54]. SOD provides the first line of defense against free radicals by making toxic superoxide into less toxic H 2 O 2 [55], while GSH and GST generally guarantee the removal of H 2 O 2 . Our experimental results confirmed the above conclusion that compared with the control group, the levels of GST (Figure 3c (Figure 3b) significantly increased. It was suggested that lead acetate caused oxidative stress and lipid peroxidation in mice. Compared with the model group, the LBP administration group significantly upregulated the levels of GST, GSH, and SOD and downregulated the level of MDA in mice with lead-induced nephrotoxicity. Experiments by Varoni et al. [56] demonstrated that LBP could inhibit the oxidative stress response and thus protect from cadmium-induced rat liver damage. The experiment of Pan et al. showed that [57] LBP can improve retinal oxidative stress and exert beneficial neuroprotective effects in diabetic rats, and its mechanism may be associated with the activation of the Nrf2/HO-1 antioxidant pathway. It is suggested that LBP may regulate the production of antioxidant enzymes against lead-induced renal injury by activating the Nrf2 signaling pathway.
Lead-induced cellular inflammation is also considered to be one of the mechanisms of renal injury [41]. Therefore, the inflammatory markers in the kidney tissue of each group of mice were detected. TNF-α and IL-1β are produced from the activated macrophages and lymphocytes; they are involved in the local and systemic inflammatory responses at the injured site [58]. Moreover, during this inflammatory response, ROS/nitrogenous species (RNS) are produced, which can lead to obvious damage to tissues and cells [35]. Our study showed that the levels of IL-1β (Figure 4b), TNF-α (Figure 4a), and NO ( Figure 4c) were increased in the model group compared with the control group. Excitingly, the LBP administration group could significantly downregulate the concentrations of TNF-α, NO, and IL-1β in lead-induced nephrotoxic mice. According to Xiong et al. [23], LBP reduced inflammation by upregulating nuclear factor erythroid-2-related factor 2 (Nrf2) and oxygenase-1 (HO-1). It is suggested that LBP may protect the kidneys of lead-induced nephrotoxic mice by inhibiting the expression of proinflammatory cytokines such as IL-1β, TNF-α, and NO and the activation of the Nrf2 pathway.
Renal apoptosis is associated with the development of oxidative stress and inflammation [59]. Therefore, apoptosis markers were detected. Our results showed that the concentrations of the Casp-3 ( Figure 5a) and Bax (Figure 5b) proteins were significantly increased, while the concentrations of Bcl-2 ( Figure 5c) protein were significantly decreased in the model group compared with the control group. This is consistent with the results of previous studies [6,52]. Bax is a principal apoptotic protein, whereas Bcl-2 is an apoptosis antagonist [60]. The best recognized biochemical hallmark of both the early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals [61]. Liu et al. suggested that lead can induce depletion of mitochondrial membrane potential and then cause apoptosis in the proximal tubules of rats [12]. Additionally, it has been reported in the literature that lead-induced toxicity is associated with infiltration of inflammatory cells in the interstitial space, which stimulates inflammationmediated apoptosis via the extrinsic pathway [62]. Strikingly, our results showed that LBP significantly downregulated Casp-3 enzyme and Bax protein concentrations, while upregulating Bcl-2 protein concentrations in the kidney tissues of lead-toxic mice. Research by Yu et al. showed [63] that LBP could protect primary cultured hippocampal neurons from oxygen-glucose deprivation/reoxygenation-induced apoptosis and autophagy cell death through phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin (mTOR) signaling pathway. Yang et al. [21] showed that LBP could promote the development of pre-cryopreserved mouse two-cell embryos by restoring the mitochondrial function and downregulating the production of reactive oxygen species. In addition, the research of Luo et al. [64] showed that LBP could prevent the decline of mitochondrial membrane potential, upregulate the expression of Bcl-2, and downregulate the expression of Bax, thereby reducing the apoptosis of spermatogenic cells. These results suggest that LBP may protect renal cells from lead-induced apoptosis by improving mitochondrial dysfunction, upregulating the expression of Bcl-2, and downregulating the expression of Bax and Casp-3.
The Nrf2 signaling pathway plays a key role in maintaining cell homeostasis under conditions of oxidative stress, inflammation, apoptosis, and cancer [65]. By analyzing the expression of oxidative stress markers, inflammatory markers, and apoptosis markers in the kidneys of mice, it is speculated that the Nrf2 signal pathway may be one of the pharmacological targets of LBP against lead-induced renal injury. Therefore, expressions of Nrf2 signaling pathway-related proteins in the kidneys of mice were detected. Under normal physiological circumstances, Nrf2 is anchored in the cytoplasm by Keap1, which as an action substrate of the Cullin 3 (Cul3)-dependent E3 ubiquitin ligase complex, could promote Nrf2 to be ubiquitinated and rapidly degraded by the proteasome [66]. However, when the cell is attacked by ROS or electrophiles, Nrf2 is dissociated from Keap1 and rapidly translocated into the nucleus. It forms a heterodimer with the small Maf protein and then combines with antioxidant response elements to transcriptively activate the expression of antioxidant factor genes such as GSH, GST, SOD, NQO1, and HO-1, regulated by Nrf2 [65]. However, this is not always the case. The experiments of Liu et al. [67] and Liu et al. [68] showed that lead inhibited the expression of Nrf2 signaling-related proteins and then induced oxidative stress, inflammation, and apoptosis. Interestingly, our experimental results are not entirely consistent with previous studies. Our results showed that lead exposure significantly downregulated Nrf2 ( Figure 6b) and keap1 (Figure 6c) expressions and significantly upregulated HO-1 (Figure 6d) expression, while NQO1 ( Figure 6e) expression was not significantly different. This indicates that lead inhibits the activation of the Nfr2 signaling pathway. It is well known that the mechanism of lead-induced renal injury is multifaceted, and pathways such as mitogen-activated protein kinase (MAPK), Akt/protein kinase B (PKB) can all regulate HO-1 expression [69]. Liu et al. [70] have found that lead-induced ROS can promote the MAPK pathway, thus promoting its downstream pathway, and inducing abnormal release of inflammatory factors, leading to inflammation and kidney damage. Therefore, we speculated that the lead-induced upregulation of HO-1 protein in this experiment might be induced by other pathways rather than by the activation of the Nrf2 signaling pathway.
Consistent with our hypothesis, LBP significantly downregulated the expression of keap1 (Figure 6c), resulting in the loss of Nrf2 control, activated Nrf2 (Figure 6b) translocation to the nucleus, and upregulated the expression of downstream antioxidant factors, thus alleviating oxidative stress, inflammation, and apoptosis of renal tissue. Notably, the decrease in keap1 in this experiment seemed to have a dose-dependent relationship with LBP, but the upregulation of Nrf2 expression by LBP was most significant in the medium-dose group. It is well known that the activation of the Nrf2 signaling pathway is multipathway [71], and the targets and efficacy of different doses of drugs are various [72]. Zheng et al.' s experiment showed [73] that LBP could mediate Nrf2 activation by the AMP-activated protein kinase (AMPK) pathway and then alleviate acute lung injury induced by hyperoxia in mice. It is suggested that LBP activates the Nrf2 signaling pathway and resists lead-induced renal damage may be mediated by multiple pathways, and the targets and efficacy of the Nrf2 signaling pathway mediated by different doses of LBP are not completely the same. In a word, LBP activates the Nrf2 signaling pathway to resist lead-induced kidney injury, which is at least related to the downregulation of keap1 by LBP, and the activation Nrf2 effect of the medium dose is the best.
Finally, the dose-effect relationship of LBP against lead-induced renal injury was analyzed by establishing PCA (Figure 7a) and OPLS-DA (Figure 7b) models and a clustering heatmap (Figure 8). The results showed that the efficacy of LBP in the medium-dose group was better than that in the high-dose group and low-dose group. As a kind of highly polar macromolecule, polysaccharides are widely considered to be poorly absorbed orally [74]. The medium dose of LBP had better efficacy against lead-induced renal injury, which may have been related to the superior bioavailability of the medium dose of LBP.

Conclusions
This study aimed to investigate the efficacy and mechanism of LBP against leadinduced renal injury. The results of the pharmacodynamics experiment showed that LBP could significantly restore the renal function and renal pathological damage of mice exposed to lead toxicity, as well as increasing the food intake of mice exposed to lead toxicity and promoting their growth. Dose effect results indicated that a medium dose of LBP was superior to high and low doses. The results of mechanistic experiments showed that LBP could attenuate oxidative stress, inflammation, and apoptosis in the kidneys of mice exposed to lead toxicity by activating the Nrf2 signaling pathway. The efficacy and mechanism of LBP against lead-induced renal injury are shown in Figure 9. and promoting their growth. Dose effect results indicated that a medium dose of LBP was superior to high and low doses. The results of mechanistic experiments showed that LBP could attenuate oxidative stress, inflammation, and apoptosis in the kidneys of mice exposed to lead toxicity by activating the Nrf2 signaling pathway. The efficacy and mechanism of LBP against lead-induced renal injury are shown in Figure 9. Institutional Review Board Statement: All the animal experiments were conducted according to the current national and international laws and recommendations, and all efforts were made to minimize suffering. Mice were cared for and treated under the guidelines established by the Chinese Council on Animal Care and approved by the Guizhou Normal University Animal Care and Use Committee.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Acknowledgments:
The authors thank Chao Zhao and Xiaojian Gong from the Key Laboratory of Mountain Environmental Information System and Ecological Environment Protection, Guizhou Normal University for their help in using the experimental equipment.

Conflicts of Interest:
The authors declare no conflict of interest. Institutional Review Board Statement: All the animal experiments were conducted according to the current national and international laws and recommendations, and all efforts were made to minimize suffering. Mice were cared for and treated under the guidelines established by the Chinese Council on Animal Care and approved by the Guizhou Normal University Animal Care and Use Committee.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.